Stroke and Traumatic Brain Injury
Markers
Alan H.B. Wu, Ph.D.
University of California, San Francisco
San Francisco General Hospital
Learning objectives
• Understand the pathophysiology of ischemic and
hemorrhagic stroke, subarachnoid hemorrhage,
and Alzheimer’s disease.
• Understand the characteristics of an ideal brainrelated biomarker.
• Describe the biomarkers for stroke, SAH, TBI,
and AD.
• Discuss current limitations of these biomarkers
• Understand the need of multi-marker analysis
Ideal characteristics for brain injury
marker: comparison to cardiac
• One biomarker rapidly increased in blood after
injury
Cardiac troponin.
• Rapid assay
• Immunoassays for Tn widely available
• Highly sensitive and specific for injury vs. mimics
Troponin not increased in other chest pain
etiologies (skeletal muscle, pancreatitis, etc).
• Differentiate between precursor disease, e.g.,
transient ischemic attack (TIAs).
Troponin increased in both AMI and UA
Ideal characteristics for brain injury
• Differentiate between ischemic vs. hemorrhagic
etiologies.
Tn increased in both STEMI & nSTEMI
• Results used for infarct sizing
Tn can be used for this.
• Results used to determine therapy.
Tn cannot be used to guide therapy
• Risk stratification for adverse outcomes
Tn effective
• Obviate the need for imaging techniques.
Does not replace ECG or echocardiography
Stroke in America
• No. 3 cause of death when considered separately from
other CV diseases (heart disease and cancer).
• 700,000 Americans experience a new or recurrent stroke
each year.
• Of all strokes, 88% are ischemic, 9% are intracerebral
hemorrhagic, and 3% are subarachnoid hemorrhage.
• Age-adjusted incidence for first stroke are (per 100,000):
167 white males
138 white females
323 black males
260 black females
Diagnostic markers for stroke
Misdiagnosis of stroke
Norris et al. Lancet 1982;1:328-31
• 821 patients consecutively admitted from
ER
• Evaluators- interns then neurology
• Initial studies- history and physical, LP,
EEG, CT
• Pre-CT misdiagnosis 13%
• Post-CT misdiagnosis 16%
• Corrected diagnoses:
– Seizures, previous stroke, dementia,
intoxication
Conditions that mimic stroke
Libman et al. Arh Neurol 1995;52:1119-22.
• Stroke mimic rate 19%
• Four conditions majority of mimics
• Unrecognized seizures/postictal
• Systemic infections
• Brain tumor
• Toxic metabolic
• 14 other diagnoses…
Current limitations in the diagnosis
of acute stroke
• CT-scan useful for hemorrhagic but not
ischemic stroke.
• CT abnormalities not observed during the initial
few hours
• Several neurologic diseases produce similar
symptoms.
• High-end imaging techniques not readily
available in the ED (e.g., transcranial doppler
echo).
• Absence of a suitable blood biomarker.
(Allder Lancet, 1999;354:1523)
Hemorrhagic stroke
Blood in
brainstem
Courtesy V. Wu, MD
Effingham, IL
NINDS time recommendations
• Door-to-MD:
10 minutes
• Door-to-Stroke
Team notification:
15 minutes
• Door-to-CT scan:
25 minutes
Window for biomarker testing
• Door-to-Drug:
(80% compliance)
60 minutes
• Door-to-Admission
3 hours
Candidate stroke markers
Maas et al. Biomark Med 2009;3:363-83
CNS isch vs. ICH early dx late dx TIA dx
size
severity
worsening
prognosis
Candidate stroke markers
Maas et al. Biomark Med 2009;3:363-83
CNS isch vs. ICH early dx late dx TIA dx
size
severity
worsening
prognosis
Candidate stroke markers
Maas et al. Biomark Med 2009;3:363-83
CNS isch vs. ICH early dx late dx TIA dx
size
severity
worsening
prognosis
Candidate stroke markers
Maas et al. Biomark Med 2009;3:363-83
CNS isch vs. ICH early dx late dx TIA dx
size
severity
worsening
prognosis
Candidate stroke markers
Maas et al. Biomark Med 2009;3:363-83
CNS isch vs. ICH early dx late dx TIA dx
size
severity
worsening
prognosis
S100
• S100: Acidic cytoplasmic calcium binding glial
protein (21 kDa) containing 2 subunits:
S100αα: heart.
S100αβ: glial cells
S100ββ: astrocytes and Schwann cells
Release of S100β
Fassbender et al. J Neurol Sci 1007;148:101
No S100β in
healthy controls
Sensitivity
(n=24)
4 h: 12%
8 h: 21%
10 h: 37%
24 h: 61%
72 h: 57%
Overall:
71%
S100β and infarct size
Missler et al. Stroke 1997;28:1956.
R=0.75
Neuron specific enolase
• 78 kDa dimeric (γγ) enzyme
• Catalyzes conversion of 2-phosphoglycerate 
phosphoenolpyruvate located in the cytoplasms
of neurons and neuroendocrine cells.
• Release into blood requires breakdown of bloodbrain barrier
Release of NSE
Fassbender et al. J Neurol Sci 1007;148:101
BNP in stroke
Nakagawa et al. Cerebrovas Dis 2005;19:157-64
EHT: essential hypertension
ICH: intracerebral hemorrhage
CI: cerebral infarction
Af: atrial fibrillation
Subarachnoid hemorrhage
arachnoid membrane
pia matter
Case report
• 45-year old female complains of severe
headache, nausea, photophobia, and neck pain.
• She waits 24 hours before coming to the ED.
• Physical examination showed retinal
hemorrhages and loss of pupillary light reflex.
• She undergoes a CT scan and lumbar puncture.
Case report
• CT scanning is gold standard but loses
sensitivity after 12 h.
Patient
Old SAH case
Xanthochromia for SAH
• Rupture of intracranial vessel  RBC into CSF
• Cleared by microglial cells
• Free hemoglobin is metabolized by heme
oxygenase 1 and biliverdin reductase  bilirubin
• Unconjugated bilirubin can remain for 2 to 3
weeks before being cleared.
Methods for detecting xanthochromia
• Visual
• Spectrophotometric
Baseline correction
Multi-wavelength
control
case
Multiwavelength correction
A1=ε1bChem + ε1bCbili + ε1bCprot
A2=ε2bChem + ε2bCbili + ε2bCprot
A3=ε3bChem + ε3bCbili + ε3bCprot
Absorbance
hemoglobin
billirubin
Case: bilirubin: 0.34 mg/dL
hemoglobin: 7.12 mg/dL
protein: 585 mg/dL
350
400
450
500
Wavelength
550
600
Clinical trial of SAH: LC vs multiwavelenth
Smith, Wu et al. Clin Chem 2013;submitted
CSF from 70 confirmed SAH and 70 non-SAH patients
BNP after subarachnoid hemorrhage
Taub, Wu et al. Neurocrit Care 2011;15:13-8
Traumatic brain injury
Prevalence of TBI
CDC data
• 1.4 million Americans sustain a traumatic brain
injury/year
• 275,000 hospitalizations, 80,000 disabilities, and
52,000 deaths.
• Iraq/Afghanistan conflicts, approximately 20% of
combat personnel suffered TBI.
Case report
• Duane Duerson played safety for the Chicago
Bears 1985 Super Bowl Team.
• Complains of headaches, blurred
vision and a deteriorating memory
• Suicide (GSW to chest) in 2011.
• Handwritten note to family: “Please, see that my
brain is given to the NFL’s brain bank.”
Case report
Autopsy of brain slices.
FDA-cleared biomarkers for TBI:
S100b, d-dimer, and MMP-9 after TBI
DeFaxio et al. World Neurosurg 2013, epub ahead of print
Admission
24-h
Poor vs. improved status.
Alzheimer’s Disease
Amyloid beta
• 36-43 amino acid peptide processed by the
amyloid precursor protein via β and γ-secretase
• Component of amyloid plaques in association
with Alzheimer's disease
• Aβ is neurotoxic.
Tau protein
• Stablizes microtubules that are abundant in the
CNS.
• Hyperphosphorylation of the tau protein can
result in the self-assembly of tangles of PTFs
implicated in Alzheimer’s.
Paired
helical
filaments
Biomarkers in Alzheimer
Multi-marker testing
Multimarker approach for neurology
biomarkers
• Allows simultaneous targeting of different
component of ischemic cascade
• Minimizes inaccuracies caused by aberrant
value of one analyte
• May allow diagnosis of different stroke subtypes
NSE, MBP, S-100β, thrombomodulin
Hill et al. CMAJ 2000;162:1139-40.
Marker
NSE
MBP
S-100 β
Thrombomodulin
≥1 marker on panel
Proportion of positives
0.89 (0.72-0.98)
0.39 (0.22-0.59)
0.32 (0.16-0.52)
0.43 (0.24-0.63)
0.93 (0.76-0.99)
NSE/carnosinase ratio
Butterworth et al. Stroke 1996;27:2064.
Urinary proteomic peptide profile
Molecular weight
Dawson et al. PLoS 2012;7(5):e35879.
Capillary electrophoresis migration time
Urinary proteomic peptide profile
Dawson et al. PLoS 2012;7(5):e35879.
Mild stroke vs. controls
Other biomarker stroke panel approaches
Clin Chem 2003;49:1733-39.
Case report
Jauch, Univ. Cincinnati
• Scenario:
– 58 year old man develops
sudden onset left sided
weakness, loss of balance and
slurred speech.
– Patient calls 911 for ambulance
90 min after symptom onset.
– Patient arrives to tertiary care
center without stroke team.
Case report
• Initial vital signs stable.
• History of hypertension and
smoking.
• Neurologic exam confusing to
emergency physician:
– Right facial droop and gaze
palsy
– Left arm and leg weakness
– Dysarthria
Case report
• CT scan and basic laboratory
values unremarkable.
• Physician contacts stroke team
via telemedicine to review
case.
• MMX result reported to
emergency physician.
10
MMX biomarker results
6.5
Patient’s MMX highly suggestive of
acute ischemic stroke
5.9
Optimal specificity
5
1.3
0
Optimal sensitivity
High sensitivity mass spectrometry
MALDI-TOF MS
(proteins)
LC-MS/MS
(small molecules)
Commercial platforms for multimarkers
Interpretative software for multimarker analysis
Hypothetical neural network for stroke diagnosis
Hidden
Inputs
Demographic
Neuronal
Inflammation
Hemostatic
Cytokine
Genetic
RNA Expression
Outputs
Ischemic stroke
Hemorrhagic stroke
Healthy
Classification and Regression Tree Analysis
Cook et al. Stat Med 2004;15:1439-53.
Physician Health Study:
92 SNPs from 56 candidate genes
related to inflammation, thrombosis
and lipid metabolism. 319 incident
cases of ischemic stroke and 2090
disease-free controls.
Summary
• Clinical evaluation and neuroimaging will remain
paramount
• Biomarker panel complements physical examination
and neuroimaging
• Wide variety of practice settings may benefit from
biomarker panel
• Early biomarker data can:
– Facilitate triage
– Promote faster throughput
– Complement other diagnostic tools
Quiz
Which single biomarker is used for stroke?
A.
B.
C.
D.
E.
S100
Neuron specific enolase
Myelin basic protein
Carnosinase
No single marker
Answer: E. A multimarker approach will likely be
the most successful.
Quiz
Which is correct concerning stroke biomarkers?
A.
B.
C.
D.
D-dimer is an example of a neuronal marker
S100b is an example of a inflammatory marker
NSE is an example of a genetic marker
Superoxide dismutase is an example of a oxidative
stress marker
E. TNF-α is an example of a hemostatic marker
Answer: D. A multi-marker approach will likely require
biomarkers from each of these pathophysiologic process.
Quiz
Which is correct regarding CSF bilirubin measurement?
A.
B.
C.
D.
E.
Assays typically used in serum are acceptable
Testing should be done within 4 h of symptoms
Presence of CSF hemoglobin also indicates an SAH
Xanthochromia is characterized by a yellow color
Bilirubin can be measured photometrically at 420 nm
Answer: D. A visual inspection of CSF is acceptable in the
absence of hemoglobin
Quiz
What is the most common etiology of subarachnoid
hemorrhage?
A.
B.
C.
D.
E.
Ruptured aneurysm
Hemorrhagic stroke
Traumatic brain injury
Multiple sclerosis
Ischemic stroke
Answer: A. A ruptured aneurysm will cause a bleed.